Li Yang, Yin Jiao, Qu Guosheng, Lv Luchao, Li Yadong, Yang Sheng, Wang Xing-Guo
Faculty of Life Sciences, Hubei University, Wuhan 430062, China.
Can J Microbiol. 2008 Sep;54(9):725-33. doi: 10.1139/w08-063.
A gene encoding a putative multicopper oxidase (MCO) was cloned from the soil bacterium Klebsiella sp. 601 and its corresponding enzyme was overexpressed in an Escherichia coli strain. Klebsiella sp. 601 MCO is composed of 536 amino acids with a molecular mass of 58.2 kDa. Theoretical calculation gave a pI value of 6.11. The amino acid sequence of Klebsiella sp. 601 MCO is strongly homologous to that of E. coli CueO with a similarity of 90% and an identity of 78%. Unlike E. coli CueO, Klebsiella sp. 601 MCO contains an extra 20 amino acids close to its C-terminus. The enzyme was purified to homogeneity by Ni-affinity chromatography. The purified enzyme was capable of using DMP (2,6-dimethoxyphenol), ABTS (2,2'-azino-bis(3-ethylbenzthiazolinesulfonic acid)), and SGZ (syringaldazine) as substrates with an optimal pH of 8.0 for DMP, 3.0 for ABTS, and 7.0 for SGZ. Klebsiella sp. 601 MCO was quite stable at pH 7.0 in which its activity was constant for 25 h without any significant change. Kinetic studies gave Km, kcat, and kcat//Km values of 0.49 mmol/L, 1.08 x 103 s-1, and 2.23 x 103 s-1 x mmol/L-1, respectively, for DMP, 5.63 mmol/L, 6.64 x 103 s-1, and 1.18 x 103 s-1 x mmol/L-1 for ABTS, and 0.023 mmol/L, 11 s-1, and 4.68 x 102 s-1 x mmol/L-1 for SGZ.
从土壤细菌克雷伯氏菌属601中克隆出一个编码假定多铜氧化酶（MCO）的基因，并在大肠杆菌菌株中对其相应的酶进行了过表达。克雷伯氏菌属601 MCO由536个氨基酸组成，分子量为58.2 kDa。理论计算得出的pI值为6.11。克雷伯氏菌属601 MCO的氨基酸序列与大肠杆菌CueO的氨基酸序列高度同源，相似度为90%，同一性为78%。与大肠杆菌CueO不同，克雷伯氏菌属601 MCO在其C末端附近含有额外的20个氨基酸。通过镍亲和层析将该酶纯化至同质。纯化后的酶能够以2,6 - 二甲氧基苯酚（DMP）、2,2'-联氮 - 双（3 - 乙基苯并噻唑啉 - 6 - 磺酸）（ABTS）和丁香醛连氮（SGZ）作为底物，对DMP的最佳pH为8.0，对ABTS为3.0，对SGZ为7.0。克雷伯氏菌属601 MCO在pH 7.0时相当稳定，其活性在25小时内保持恒定，没有任何显著变化。动力学研究得出，对于DMP，Km、kcat和kcat/Km值分别为0.49 mmol/L、1.08×10³ s⁻¹和2.23×10³ s⁻¹×mmol/L⁻¹；对于ABTS，分别为5.63 mmol/L、6.64×10³ s⁻¹和1.18×10³ s⁻¹×mmol/L⁻¹；对于SGZ，分别为0.023 mmol/L、11 s⁻¹和4.68×10² s⁻¹×mmol/L⁻¹。
